Biotransformation of jervine by Cunninghamella echinulata

Article abstract of DOI:10.1002/hlca.201200345

Biotransformation of jervine (1) by Cunninghamella echinulata (ACCC 30369) was carried out. Four biotransformation products were obtained, and three of them, 3-5, were identified as new compounds. On the basis of their NMR and mass-spectral data, their st

Full text of DOI:10.1002/hlca.201200345

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Helvetica Chimica Acta – Vol. 96 (2013)
Biotransformation of Jervine by Cunninghamella echinulata
by Ke-Yu Chena), Yi-Feng Lꢀa)b), Wei-Dong Zhanga)c), Jian Tangc), Run-Hui Liua), and Hui-Liang
Li*a)
a) Department of Phytochemistry, School of Pharmacy, Second Military Medical University, No. 325
Guohe Road, Shanghai 200433, P. R. China (phone/fax: þ 86-21-81871245;
e-mail: faranli@hotmail.com)
b) School of Traditional Chinese Materia Medica 49#, Shenyang Pharmaceutical University,
Wenhua Road 103, Shenyang 110016, P. R. China
c) School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, P. R. China
Biotransformation of jervine (1) by Cunninghamella echinulata (ACCC 30369) was carried out. Four
biotransformation products were obtained, and three of them, 3 – 5, were identified as new compounds.
On the basis of their NMR and mass-spectral data, their structures were characterized as jervinone (2),
7a-hydroxyjervine (3), 14a-hydroxyjervine (4), and 1b,7a-dihydroxyjervine (5). The X-ray diffraction
structure of 1 is also reported for the first time.
Introduction. – Microbial transformation is an effective method for the structural
modification of bioactive natural compounds. Its application in asymmetric synthesis is
increasing due to its versatility and ease [1]. A variety of transformations on natural
products such as oxidation, reduction, hydrolysis, isomerization, epimerization,
rearrangement, D-homoannulation, Michael addition, and reverse aldol reaction have
been already performed [2]. Cunninghamella echinulata, a filamentous fungus, is
recognized for its potential for steroid hydroxylation and has been noted for its ability
to mimic mammalian hepatic metabolism with other substrates [3][4].
Veratrum alkaloids are a group of potent hypotensive agents that act by reflex
suppression of the cardiovascular system [5][6]. Jervine (1) is one of the most
important steroidal alkaloid derived from the Veratrum genus. Similar to cyclopamine,
which also occurs in the Veratrum genus, it is a teratogen implicated in birth defects
when consumed by animals during a certain period of their gestation [7 – 10].
Biotransformations of Veratrum alkaloids, such as rubijervine, jervine, and veratramine
with various microorganisms have been reported previously [11 – 13].
In continuation of our studies on the phytochemistry of Veratrum alkaloids [14][15]
and biotransformation studies on the Veratrum alkaloids [16], we now report the
characterization of four metabolites of jervine (1) in cell suspension of C. echinulata
(ACCC 30369). These metabolites were identified as jervinone (2), 7a-hydroxyjervine
(3), 14a-hydroxyjervine (4) and 1b,7a-dihydroxyjervine (5) by spectroscopic methods
(Fig. 1). We also report here the X-ray diffraction structure of jervine (1) for the first
time.
Results and Discussion. – Compound 3 was obtained as a white amorphous powder,
showing a positive reaction with Dragendorffꢀs reagent. The HR-ESI-MS (positive-ion
ꢁ 2013 Verlag Helvetica Chimica Acta AG, Zꢂrich

Helvetica Chimica Acta – Vol. 96 (2013)
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Fig. 1. Structures of compounds 1 – 5
mode) provided the molecular formula C27H39NO4 m/z 442.2952 ([M þ H]þ). The IR
spectrum exhibited absorptions for an OH group (3370 and 1070 cmꢀ1). The 13C-NMR
spectrum (Table 1) of 3 showed signals due to four Me, seven CH2, ten CH groups, and
six quaternary C-atoms, including one C¼C bond (d(C) 142.3 (C(5)), 123.4 (C(6))),
and three O-bearing sp3-C-atoms. The 1H-NMR spectrum (Table 2) displayed
resonances for four Me groups (d(H) 0.85 (d, J ¼ 6.6, Me(27)), 0.89 (s, Me(19)), 0.90
(d, J ¼ 7.1, Me(21)), 2.01 (s, Me(18)), and the 13C-NMR exhibited the signals of two C-
atoms attached to an N-atom at d(C) 65.4 (C(22)) and 56.7 (C(26)), which are
characteristic for a jervine-type steroid alkaloid. The OH is located at C(7) due to the
HMBCs between the H-atom resonating at d(H) 4.61 – 4.67 (m, HꢀC(7)) and C(8)
(d(C) 37.0). Furthermore, the relative configuration of 7-OH was deduced as a by
NOESY correlation between HꢀC(7) and the H-atom resonating at d(H) 2.30 – 2.36
(m, HbꢀC(8)). Therefore, the structure of 3 was elucidated as 7a-hydroxyjervine.
Table 1. 13C-NMR Data of 3 – 5. At 125 MHz in CD3OD; d in ppm. Atom numbering as indicated in
Fig. 1.
C-Atom
3
4
5
C-Atom
3
4
5
C(1)
38.2
29.6
67.9
31.1
142.3
123.4
68.8
37.0
61.6
34.8
38.1
29.5
68.1
30.8
141.9
122.3
32.6
36.8
61.9
34.9
74.3
29.5
67.2
31.3
143.2
124.5
69.5
38.2
61.7
34.8
C(15)
C(16)
C(17)
C(18)
C(19)
C(20)
C(21)
C(22)
C(23)
C(24)
C(25)
C(26)
C(27)
24.3
28.4
84.3
11.6
18.1
39.8
10.6
65.4
76.0
38.2
31.3
56.7
18.7
24.1
28.7
84.3
11.6
18.5
39.8
10.7
65.8
75.2
38.5
31.3
56.6
18.9
24.0
28.8
84.7
11.7
18.0
39.6
10.7
65.9
75.4
38.5
31.5
56.8
19.1
C(2)
C(3)
C(4)
C(5)
C(6)
C(7)
C(8)
C(9)
C(10)
C(11)
C(12)
C(13)
C(14)
206.3
138.5
147.1
46.3
206.9
138.4
146.5
77.8
206.6
138.4
146.7
45.5
Compound 4 was obtained as a white amorphous powder, showing a positive
reaction with Dragendorffꢀs reagent. The HR-ESI-MS (positive-ion mode) gave the

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Helvetica Chimica Acta – Vol. 96 (2013)
molecular formula C27H39NO4 m/z 442.2956 ([M þ H]þ). The IR spectrum exhibited
absorptions for OH groups (3361 and 1073 cmꢀ1). The 13C-NMR data of 4 were similar
to those of jervine (1) except for the presence of a OH group. Therefore, 4 was
determined as an oxidized analog of 1. The OH was localized at C(14) due to the
HMBCs between H-atom resonating at d(H) 1.72 – 1.77 (m, 1 H, CH2(15)) and C(14)
(d(C) 77.8). Comparing the spectra of 4 with those of compound 1, the chemical shifts
of C(8), C(12), C(13), and C(15) were similar to those found in compounds 1, 3, and 5,
indicating the relative a-configuration for 14-OH. Thus, the structure of 4 was
identified as 14a-hydroxyjervine.
Compound 5 was obtained as a white amorphous powder, showing a positive
reaction with Dragendorffꢀs reagent. The HR-ESI-MS (positive-ion mode) led to the
molecular formula C27H39NO5 (m/z 458.2906, [M þ H]þ). The IR spectrum exhibited
absorptions for OH groups (3361 and 1068 cmꢀ1) and C¼C bonds (1624 cmꢀ1). The 13C-
(Table 1) and 1H-NMR (Table 2), and ESI mass spectra suggested it to be a
dihydroxylated jervine derivative. Three OH groups are localized at C(1) (d(C)
74.3), C(3) (67.2), and C(7) (69.5), respectively, due to the HMBC correlations
between the H-atom resonating at d(H) 3.85 – 3.89 (m, HꢀC(1)) and C(2) (d(C) 29.5),
the H-atom resonating at d(H) 3.86 – 3.92 (m, HꢀC(3)) and C(2) (d(C) 29.5), C(4)
(31.3), and the H-atom resonating at d(H) 4.59 – 4.66 (m, HꢀC(7)) and C(6) (d(C)
124.5), C(8) (38.2). The stereochemical relationship among the functional groups, i.e.,
3-OH, Me(19), Me(21), and Me(27), was established to be identical to that of the
Table 2. 1H-NMR Data of 3 – 5. At 500 MHz in CD3OD; d in ppm, J in Hz. Atom numbering as indicated in
Fig. 1.
H-Atom
3
4
5
CH2(1)
CH2(2)
HꢀC(3)
CH2(4)
HꢀC(6)
HꢀC(7)
HꢀC(8)
HꢀC(9)
2.51 – 2.53 (m), 2.12 – 2.17 (m) 2.47 – 2.52 (m), 2.10 – 2.13 (m) 3.85 – 3.89 (m)
1.80 – 1.85 (m), 1.70 – 1.74 (m)
4.12 – 4.21 (br. s)
1.80 – 1.84 (m), 1.70 – 1.75 (m)
4.11 – 4.26 (m)
2.29 – 2.34 (m), 1.92 – 1.96 (m)
3.86 – 3.92 (m)
2.41 – 2.47 (m), 1.52 – 1.61 (m) 2.38 – 2.45 (m), 1.51 – 1.55 (m) 2.57 – 2.63 (m), 2.39 – 2.44 (m)
5.35 (d, J ¼ 5.1)
4.61 – 4.67 (m)
2.30 – 2.36 (m)
1.90 – 1.97 (m)
5.35 (d, J ¼ 5.1)
5.62 (d, J ¼ 5.1)
2.21 – 2.27 (m), 2.05 – 2.09 (m) 4.59 – 4.66 (m)
1.63 – 1.67 (m)
1.81 – 1.85 (m)
–
2.35 – 2.39 (m)
2.04 – 2.07 (m)
2.09 – 2.13 (m)
HꢀC(14) 1.96 – 2.01 (m)
CH2(15)
CH2(16)
Me(18)
Me(19)
1.92 – 1.97 (m), 1.31 – 1.33 (m)
2.21 – 2.24 (m), 1.72 – 1.77 (m) 1.99 – 2.04 (m), 1.39 – 1.42 (m)
2.33 – 2.36 (m), 1.38 – 1.41 (m) 2.07 – 2.11 (m), 1.68 – 1.71 (m) 2.33 – 2.36 (m), 1.32 – 1.36 (m)
2.01 (s)
0.89 (s)
2.04 (s)
0.85 (s)
2.01 (s)
0.91 (s)
HꢀC(20) 2.53 – 2.57 (m)
2.57 – 2.62 (m)
0.91 (d, J ¼ 7.0)
2.64 – 2.69 (m)
3.66 – 3.69 (m)
2.55 – 2.61 (m)
0.91 (d, J ¼ 7.0)
2.67 – 2.70 (m)
3.62 – 3.66 (m)
Me(21)
0.90 (d, J ¼ 7.1)
HꢀC(22) 2.68 – 2.71 (m)
CH2(23)
CH2(24)
3.43 – 3.50 (m)
2.26 – 2.30 (m), 1.17 – 1.22 (m) 2.23 – 2.26 (m), 1.13 – 1.16 (m) 2.24 – 2.27 (m), 1.09 – 1.13 (m)
HꢀC(25) 1.48 – 1.53 (m)
1.49 – 1.52 (m)
1.47 – 1.52 (m)
CH2(26)
3.14 – 3.18 (dd, J ¼ 12.0, 3.5),
3.07 – 3.11 (dd, J ¼ 11.0, 3.5),
3.19 – 3.25 (dd, J ¼ 12.0, 6.0),
2.34 – 2.38 (m)
2.28 – 2.31 (m)
2.29 – 2.34 (m)
Me(27)
0.85 (d, J ¼ 6.6)
0.87 (d, J ¼ 6.6)
0.86 (d, J ¼ 6.0)

Helvetica Chimica Acta – Vol. 96 (2013)
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starting material 1 by NOESY analysis, and also by comparison of chemical shifts and
coupling constants of 5 with those of 1. The relative configuration of 1-OH and 7-OH
was deduced as b/a, respectively, by the NOESY correlations between HꢀC(1)
resonating at d(H) 2.04 – 2.07 (m, HaꢀC(9)) and HꢀC(7) resonating at 2.35 – 2.39 (m,
HbꢀC(8)) (Fig. 2). Therefore, the structure of 5 was identified as 1b,7a-dihydrox-
yjervine.
Fig. 2. Key NOESY correlations of 5
This work was supported by the Program NCET Foundation, NSFC (30725045, 81102335), partially
supported by Global Research Network for Medicinal Plants (GRNMP) and King Saud University,
Shanghai Leading Academic Discipline Project (B906), FP7-PEOPLE-IRSES-2008 (TCMCANCER
Project 230232), Key Laboratory of Drug Research for Special Environments, PLA, Shanghai
Engineering Research Center for the Preparation of Bioactive Natural Products (10DZ2251300), and
the Scientific Foundation of Shanghai China (09DZ1975700, 09DZ1971500,10DZ1971700, and
11QA1408200).
Experimental Part
General. Column chromatography (CC): MCI gel CHP20P (high-porous polymer 75 – 150 mm;
Mitsubishi Chemicals, Japan), reversed-phase (RP) silica gel RP-18 (40 – 63 mm; Merck, Germany), silica
gel (SiO2; 200 – 300 mesh; Yantai, P. R. China), and Sephadex LH-20 (Pharmacia Co., Ltd.). TLC: SiO2
plates; visualization by spraying with 10% H2SO4 in EtOH, followed by heating. Optical rotation: Perkin-
Elmer 341 polarimer. IR Spectra: Nicolet-Compact-40-Bruker-Vector-22 spectrophotometer; ˜n in cmꢀ1
.
NMR Spectra: Bruker AVANCEII-500 NMR, for 1H at 500 and 13C at 125 MHz; d in ppm rel. to Me4Si as
internal standard, J in Hz. MS: Varian MAT-212 mass spectrometer (for ESI) and Q-Tof micro mass
spectrometer (for HR-ESI); in m/z.
Plant Material. The plants were collected in Yanbian, Jilin Province, P. R. China, in September 2005,
and authenticated as V. dahuricum by Prof. Yong-Zhen Liu, School of Pharmacy, Yanbian University,
China. A voucher specimen (No. 211) was deposited with the Herbarium of the School of Pharmacy,
Shanghai Jiao Tong University, Shanghai, China.
Organisms. The stock culture of C. echinulata (ACCC 30369) was maintained on a potato dextrose
agar slant. Four 1000-ml Erlenmeyer flasks, each containing 300 ml of liquid medium consisting of 0.1%
peptone, 0.1% yeast extract, 0.1% beef extract, and 0.5% glucose were inoculated with freshly obtained
C. echinulata cultured from the agar slant on a rotary shaker at 180 rpm. After cultivation at 288 for 72 h,

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Helvetica Chimica Acta – Vol. 96 (2013)
the jervine (1) soln. (50 mg of 1 dissolved in 250 ml of EtOH) was added to each flask, and the incubation
was continued for 12 d.
Microbial Metabolism of Jervine (1). After 12 d of incubation, the incubation mixtures were pooled
and filtered. The filtrate was passed through a MCI-gel CHP20P column, and washed with H2O to
remove sugars; then, successive extraction with 20% aq. MeOH, 60% aq. MeOH, and MeOH gave the
Fractions A, B, and C, resp. Each fraction was evaporated to dryness in vacuo and analyzed by TLC
(CHCl3/MeOH/H2O 65 :35 :10; visualization by spraying with 10% H2SO4 soln.). Fr. C was chromato-
graphed repeatedly through a column on SiO2 (CHCl3/MeOH 10 :1) to afford 2 (8.8 mg). Fr. B was
subjected repeatedly to CC (SiO2; CHCl3/MeOH 5 :1; Sephadex LH-20; 70% MeOH) to furnish 3
(4.6 mg) and 4 (6.1 mg). Fr. A was dissolved in 10% aq. MeOH and subjected to CC (RP SiO2 (ODS);
H2O/MeOH 100 :10 ! 0 :100) to give 5 (3.6 mg).
Jervine (1). Colorless prisms. C27H39NO3. ESI-MS: 426 ([M þ H]þ). [a]D23 ¼ ꢀ58.2 (c ¼ 3.08,
MeOH). Jervine (1) was isolated from the roots of Veratrum dahuricum with purity of 99.0% by HPLC
analysis in our laboratory. The chemical structure was identified by detailed NMR analysis and
comparing its data with those in the literature [17]. An X-ray diffraction analysis of (ꢀ)-varatrobasine
(¼ jervine-11b-ol) has been reported previously [18]. We performed the X-ray-analysis of 1 (Fig. 3) to
establish the absolute configuration of (ꢀ)-jervine.
Fig. 3. X-Ray crystal structure of 1
7a-Hydroxyjervine (¼(3b,7a,22S,23R)-3,7-Dihydroxy-17,23-epoxyveratraman-11-one ¼ (3S,3’R,
3a’S,6S,6’S,6aS,6bS,7a’R,9R,11aS,11bR)-2,3,3’a,4,4’,5’,6,6’,6a,6b,7,7’,7’a,8,11a,11b-Hexadecahydro-3,6-di-
hydroxy-3’,6’,10,11b-tetramethylspiro[9H-benzo[a]fluorene-9,2’(3’H)-furo[3,2-b]pyridin]-11(1H)-one;
3). White amorphous powder. [a]2D3 ¼ ꢀ113.4 (c ¼ 0.81, MeOH). IR (KBr): 3370, 2951, 2177, 1623, 1070.
1H- and 13C-NMR: Tables 2 and 1, resp. 2D-NMR (HMBC; 500 MHz, CD3OD): HꢀC(3)/C(4), HꢀC(7)/
C(5), HꢀC(7)/C(8), HꢀC(15)/C(14). ESI-MS: 442 ([M þ H]þ). HR-ESI-MS: 442.2952 ([M þ H]þ,
C27H40NOþ4 ; calc. 442.2957).
14a-Hydroxyjervine (¼(3b,22S,23R)-3,14-Dihydroxy-17,23-epoxyveratraman-11-one ¼ (3S,3’R,
3a’S,6’S,6aR,6bR,7a’R,9R,11aS,11bR)-2,3,3’a,4,4’,5’,6,6’,6a,6b,7,7’,7’a,8,11a,11b-Hexadecahydro-3,6b-di-
hydroxy-3’,6’,10,11b-tetramethylspiro[9H-benzo[a]fluorene-9,2’(3’H)-furo[3,2-b]pyridin]-11(1H)-one;
4). White amorphous powder. [a]2D3 ¼ ꢀ54.6 (c ¼ 0.76, MeOH). IR (KBr): 3361, 2947, 2170, 1617, 1073.
1H- and 13C-NMR: Tables 2 and 1, resp. 2D-NMR (HMBC; 500 MHz, CD3OD): HꢀC(3)/C(4), HꢀC(7)/
C(8), HꢀC(15)/C(14). ESI-MS: 442 ([M þ H]þ). HR-ESI-MS: 442.2956 ([M þ H]þ, C27H40NOþ4 ; calc.
442.2957).
1b,7a-Dihydroxyjervine (¼(1b,3b,7a,22S,23R)-1,3,7-Trihydroxy-17,23-epoxyveratraman-11-one ¼
(1R,3R,3’R,3a’S,6S,6’S,6aS,6bS,7a’R,9R,11aS,11bR)-2,3,3’a,4,4’,5’,6,6’,6a,6b,7,7’,7’a,8,11a,11b-Hexadeca-

Helvetica Chimica Acta – Vol. 96 (2013)
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hydro-1,3,6-trihydroxy-3’,6’,10,11b-tetramethylspiro[9H-benzo[a]fluorene-9,2’(3’H)-furo[3,2-b]pyridin]-
11(1H)-one; 5). White amorphous powder. [a]D23 ¼ ꢀ8.9 (c ¼ 0.60, MeOH). IR (KBr): 3361, 2937, 2155,
1
1624, 1068. H- and 13C-NMR: Tables 2 and 1, resp. 2D-NMR (HMBC; 500 MHz, CD3OD): HꢀC(1)/
C(2), HꢀC(3)/C(4), HꢀC(7)/C(5), HꢀC(7)/C(8). ESI-MS: 458 ([M þ H]þ). HR-ESI-TOF-MS:
458.3304 ([M þ H]þ, C27H40NOþ5 ; calc. 458.2906).
X-Ray Diffraction Structure Analysis of Jervine1). Single crystal for analysis was obtained from
CHCl3/MeOH/H2O. Data collection was performed with a Bruker APEX2 CCD and graphite
monochromated CuKa radiation (l ¼ 1.54178 ꢃ) at 133(2) K. Crystallgraphic data: C27H39NO3 ·
CH3Cl · 2 H2O; Mr 512.11; crystal size 0.25 ꢁ 0.20 ꢁ 0.18 mm, orthorhombic, space group P212121; a ¼
7.4412(10) ꢃ, b ¼ 9.9446(10) ꢃ, c ¼ 36.9172(3) ꢃ, a ¼ b ¼ g ¼ 908; V¼ 2731.86(5) ꢃ3 ; Z ¼ 4; Dx ¼
1.245 g cmꢀ3; F000 ¼ 1112; m(CuKa) 1.535 mmꢀ1. Cell refinement and data reduction: Bruker SAINT.
Program used to solve and refine structure: SHELXS-97 and SHELXL-97, resp.; refinement on F2 with
full-matrix least-squares calculations. All non-H-atoms were filtered with anisotropic parameters, and all
H-atoms were positioned by geometrical calculation and refined by ride-on method with relative
isotropic parameters. Absorption correction were applied with semi-empirical from equivalents (maxmin
transmission, 0.7679/0.7003). 12357 Reflections (4615 unique, Rint ¼ 0.0218) were measured in the q range
of 2.39 – 66.568; limiting indices, 8 ꢂ h ꢂꢀ 8, 11 ꢂ k ꢂꢀ 11, 41 ꢂ l ꢂꢀ 43. The final phase converged to
R1 ¼ 0.0289 (wR2 ¼ 0.0787) for 4615 observed reflections (I > 2s(I)) and 331 refined parameters, R1 ¼
0.0294 (wR2 ¼ 0.0790) for all unique reflections; goodness-of-fit on F2, 1.046.
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Received July 4, 2012
1
)
CCDC-889549 contains the supplementary crystallographic data for this article. These data can be
obtained free of charge via http://www.ccdc.cam.ac.uk/data_request/cif.